CN102998155A - Device and method for online deduction of amino acid disulfide and determination of amino acid - Google Patents
Device and method for online deduction of amino acid disulfide and determination of amino acid Download PDFInfo
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- CN102998155A CN102998155A CN2012104831363A CN201210483136A CN102998155A CN 102998155 A CN102998155 A CN 102998155A CN 2012104831363 A CN2012104831363 A CN 2012104831363A CN 201210483136 A CN201210483136 A CN 201210483136A CN 102998155 A CN102998155 A CN 102998155A
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Abstract
The invention provides a device and a method for online deduction of amino acid disulfide and determination of amino acid. The device mainly comprises a high-pressure pump, a high performance liquid chromatographic column, an online reduction column, and a chemiluminescent detector. The basic technical route is as follows: a amino acid solution to be determined enters the online reduction column after being subjected to high performance liquid chromatographic column separation; and the amino acid disulfide in the solution is restored into corresponding mercaptoamino acid by tri(2-propyloic) phosphine complex crystal of zinc (II) in the online reduction column, and then enters the chemiluminescent detector to be subjected to online determination of the amino acid. The online reduction device is simple and convenient in detection method, rapid in reduction process, high in reduction efficiency, good in repeatability, and wide in application prospect, and newly restored mercaptoamino acid is prevented from being oxidized into the amino acid disulfide again in sample treatment and analysis processes.
Description
Technical field
The present invention relates to a kind of fast online reducing amino acids disulfide and measure amino acid whose device and method, this device and method is applicable to the content of mercaptoamino acid and amino acid disulfide in online Fast Measurement urine or the plasma sample.
Background technology
The content of mercaptoamino acid and amino acid disulfide and human body is healthy closely bound up in the biofluid.By the mensuration to mercaptoamino acid and amino acid disulfide content, health that can the Real Time Monitoring human body.Used method is by off-line amino acid disulfide wherein to be reduced into corresponding mercaptoamino acid at present, and then measures the content of various mercaptoamino acids, thereby determines various amino acid whose content in the sample.Document Muhammad I.K., Zafar I.Simultaneous determination ofascorbic acid, aminothiols, and methionine in biological matrices using ion-pairing RP-HPLCcoupled with electrochemical detector.J.Chromatogr., B, 2009,877:2567 – 2575. has introduced the method.This off-line operation is not only lost time, and mercaptoamino acid is easily become amino acid disulfide by autoxidation in sample preparation and ensuing analytic process.Document James L., James D.The determination of disulphide species withinphysiological fluids.Trends Anal.Chem., 2012,21:807-815. reported employing galvanochemistry or mass spectrometry method directly while on-line determination mercaptoamino acid and amino acid disulfide, fairly simple on this assay method program, but the electrochemical method electrode stability is poor, easily is disturbed the material impact, causes sensitivity; Mass spectrometry method equipment is complicated, and expensive.Most mensuration mercaptoamino acids and amino acid disulfide still adopt indirect method at present, and these method operation stepss are many, relatively lose time.Therefore, need a kind of fast simple online method of reducing and measuring simultaneously amino acid disulfide and mercaptoamino acid of design, by piece-rate system, online restoring system and mensuration system are coupled together, reach the purpose of measuring simultaneously mercaptoamino acid and amino acid disulfide.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of fast online reducing amino acids disulfide be provided and measure amino acid whose device and method,
Online reducing amino acids disulfide provided by the present invention is also measured amino acid whose device as shown in Figure 1, wherein mainly is comprised of high-pressure pump, high performance liquid chromatography separating column, online reduction column, chemiluminescence detector; Wherein high-pressure pump (1a) is connected with the reduction column of being connected (4) with high performance liquid chromatography separating column (3) successively through six-way injection valve (2), is connected with chemiluminescence detector (5) again.Pump (1b), pump (1c), pump (1d) are arranged successively by T-shaped connecting pipe access between online reduction column (4) and the chemiluminescence detector (5).
Structure such as Fig. 2 of described online reduction column (4), consisted of by pitch tube (1), glass fibre (2), TCEP Complex Crystal (3) and quartz ampoule (4), wherein TCEP Complex Crystal (3) is filled in the quartz ampoule (4), two ends all are filled with glass fibre (2), and the two ends of quartz ampoule (4) use respectively pitch tube (1) to be connected with other instruments.
Described quartz ampoule length is 4-12cm, and internal diameter is 1-3mm; Quartz ampoule is clean with pure water rinsing before use, in 100 ℃ of baking ovens dry 2-3 hour; Reduction column will be used pure water rinsing 3 hours before use.
Described TCEP Complex Crystal is three (2-carboxyethyl) phosphine Complex Crystal of zinc (II).
Utilize this device to carry out online reducing amino acids disulfide and measure amino acid whose assay method to be: start simultaneously 4 high-pressure pumps (1a), (1b), (1c), (1d) respectively with trifluoroacetic acid solution, triangle gold nano solution, luminol solution, superoxol injected system simultaneously; When wherein trifluoroacetic acid solution flows through six-way injection valve (2), to be written into high performance liquid chromatography separating column (3) by the testing sample that syringe (6) are injected in the six-way injection valve (2), enter online reduction column (4) after each seed amino acid in the sample is separated, amino acid disulfide in the solution is reduced into corresponding mercaptoamino acid by the TCEP complex compound, again successively with triangle gold nano solution, luminol solution, superoxol on-line mixing, enter at last chemiluminescence detector (5) and carry out on-line determination.
Described testing sample is human plasma or urine, wherein contained amino acid is the potpourri of mercaptoamino acid and amino acid disulfide, and wherein mercaptoamino acid is the potpourri of halfcystine, cysteinyl glycine, homocysteine, glutathione and glutamyl cysteine; Amino acid disulfide is the potpourri of cystine and homocystine.
Described triangle gold nano solution is according to document Q.Q.Li et al.Aminothiols Sensing Based onFluorosurfactant-Mediated Triangular Gold Nanoparticle-Catalyzed Luminol Chemiluminescence.J.Phys.Chem.C, 2011,115:10964-10970. the method for report is synthesized, triangle gold nano solution concentration is 0.2-1.06nmol/L.
The trifluoroacetic acid percent by volume is 0.05-0.25% in the described trifluoroacetic acid solution,, the luminol solution volumetric molar concentration is that 0.002-0.02mmol/L, superoxol volumetric molar concentration are 0.2-10mmol/L.
Described trifluoroacetic acid solution, triangle gold nano solution, luminol solution, superoxol flow velocity are 0.2-2mL/min.
Reduction process in the online reduction column is
[NH
4][Zn(TCEP)]+RSSR+H
2O→[NH
4][Zn(TCEPO)]+2RSH
Wherein RSSR is cystine or homocystine, and RSH is halfcystine or homocysteine.
The amino acid disulfide solution is reduced into mercaptoamino acid after flowing through online reduction column.Only have mercaptoamino acid to be measured to by chemiluminescence detector.
Described TCEP Complex Crystal is according to document Artur K.et al.Coordination Properties ofTris (2-carboxyethyl) phosphine, a Newly Introduced Thiol Reductant, and Its Oxide.Inorg.Chem., the method of 2003,42:1994-2003. report is synthesized.
When online reduction column does not use for a long time, be placed in 4 ℃ the refrigerator and preserve.
The online reduction column device that amino acid disulfide in the biofluid is reduced to corresponding mercaptoamino acid provided by the invention, has manufacturing process simple, reduction process is quick, easy, reduction efficiency is high, good reproducibility, avoid the mercaptoamino acid of new reduction in sample preparation and analytic process, again to be oxidized to the advantages such as amino acid disulfide, had higher actual application prospect.
Description of drawings
Fig. 1 is the structural representation of online reduction column and high performance liquid chromatography coupling.Among the figure: 1-high-pressure pump, 2-six-way injection valve, 3-chromatographic column, the online reduction column of 4-, 5-chemiluminescence detector, 6-syringe.
Fig. 2 is the structural representation of online reduction column.Among the figure: 1-pitch tube, 2-glass fibre, 3-TCEP Complex Crystal, 4-quartz ampoule.
Fig. 3 is the chromatogram of online determination by reduction of the present invention and the chromatogram contrast of non-online reduction, and the chromatogram of the online reduction of A right and wrong, B are the chromatograms of online reduction; Peak sequence is: 1, cystine, 2, halfcystine, 3, cysteinyl glycine, 4, homocysteine, 5, homocystine, 6, glutathione, 7, glutamyl cysteine.
Embodiment
Online reducing amino acids disulfide provided by the present invention is also measured amino acid whose device as shown in Figure 1, the model of used high-pressure pump is LC-15C, six-way injection valve is Luo Danni 7725i, the high performance liquid chromatography separating column is the C-18 post, and chemiluminescence detector is ultraweak chemical luminescence detector.
Structure such as Fig. 2 of described online reduction column (4), wherein quartz ampoule length is 10cm, and internal diameter is 2mm, and is clean with pure water rinsing before use, and drying is 3 hours in 100 ℃ of baking ovens; Online reduction column (4) is used pure water rinsing 3 hours before use.
Described TCEP Complex Crystal is three (2-carboxyethyl) phosphine Complex Crystal of zinc (II), synthesizes in accordance with the following steps:
Be that the perchloric acid solution of 3mol/L slowly is added drop-wise in the 100mg zinc paste with volumetric molar concentration, until zinc paste all dissolves.Add again the 5mL absolute ethyl alcohol, then be heated to 50 ℃, absolute ethyl alcohol is slowly volatilized, obtain at last six perchloric acid hydrate zinc crystal.Carry out again recrystallization one time, then in a vacuum 50 ℃ of oven dry.
0.2mmol three (2-carboxyethyl) phosphonium salt hydrochlorate l and 0.2mmol six perchloric acid hydrate zinc are dissolved in water/methanol solution of 5mL 1:3, add again the ammoniacal liquor of 1.5mmol, stirred standing over night 10 minutes.Water/methanol solution and the deionized water of sediment with 1:3 washed respectively 3 times, and 50 ℃ of oven dry obtain the TCEP Complex Crystal in a vacuum.
Online reducing amino acids disulfide and to measure amino acid whose concrete steps as follows:
A. halfcystine, cysteinyl glycine, homocysteine, glutathione and the glutamyl cysteine that volumetric molar concentration is 0.5 μ mol/L and volumetric molar concentration are the cystine of 0.25 μ mol/L and homocystine mixes and is made into testing sample, and simulation human urine or blood constituent carry out following mensuration.
B. start simultaneously 4 high-pressure pumps (1a), (1b), (1c), (1d) respectively with trifluoroacetic acid solution, triangle gold nano solution, luminol solution, superoxol with the flow velocity of 1mL/min injected system simultaneously; Wherein trifluoroacetic acid solution flows through six-way injection valve (2) and carries the sample that is injected by syringe and enter high performance liquid chromatography separating column (3), enter online reduction column (4) after each seed amino acid is separated, amino acid disulfide wherein is reduced into mercaptoamino acid, again successively with triangle gold nano solution, luminol solution, superoxol on-line mixing, enter at last the on-line determination that chemiluminescence detector (5) carries out mercaptoamino acid;
Wherein the percent by volume of trifluoroacetic acid solution is 0.15%, and the volumetric molar concentration of triangle gold nano solution, luminol solution, superoxol is 0.35nmol/L, 0.01mmol/L, 8mmol/L successively.
Non-online determination by reduction is carried out in contrast, method be among the above-mentioned step B each seed amino acid separated after, directly mix with the superoxol of the luminol solution of the triangle gold nano solution of 0.35nmol/L, 0.01mmol/L, the 8mmol/L flow velocity with 1mL/min, enter at last the on-line determination that chemiluminescence detector (5) carries out mercaptoamino acid.The result is shown in the A figure of Fig. 3.
Contrast by A figure and two spectrograms of B figure can be found out: non-online method of reducing can only be measured to mercaptoamino acid.And by online reduction, the amino acid disulfide that can be measured to simultaneously mercaptoamino acid and be reduced.The amino acid disulfide of 0.25 μ mol/L is reduced into behind the corresponding mercaptoamino acid unanimously with the signal of the mercaptoamino acid of 0.5 μ mol/L among the B figure, and this has illustrated that online reduction column can be reduced to mercaptoamino acid with amino acid disulfide fully.So device of the present invention can be realized online reduction and measure simultaneously amino acid disulfide and sulfhydryl amino acid content.
Claims (2)
1. an online reducing amino acids disulfide and measure amino acid whose device mainly is comprised of high-pressure pump, high performance liquid chromatography separating column, online reduction column, chemiluminescence detector; Wherein high-pressure pump (1a) is connected with the reduction column of being connected (4) with high performance liquid chromatography separating column (3) successively through six-way injection valve (2), is connected with chemiluminescence detector (5) again; High-pressure pump (1b, 1c, 1d) is arranged successively by T-shaped connecting pipe access between online reduction column (4) and the chemiluminescence detector (5);
The structure of described online reduction column (4) is made of pitch tube (1), glass fibre (2), TCEP Complex Crystal (3) and quartz ampoule (4), wherein TCEP Complex Crystal (3) is filled in the quartz ampoule (4), two ends all are filled with glass fibre (2), and the two ends of quartz ampoule (4) use respectively pitch tube (1) to be connected with other instruments;
Described quartz ampoule length is 4-12cm, and internal diameter is 1-3mm; Described TCEP Complex Crystal is three (2-carboxyethyl) phosphine Complex Crystal of zinc (II).
2. an online reducing amino acids disulfide as claimed in claim 1 and measure the assay method of amino acid whose device,
Start simultaneously 4 high-pressure pumps (1a), (1b), (1c), (1d) respectively with trifluoroacetic acid solution, triangle gold nano solution, luminol solution, superoxol while injected system; When wherein trifluoroacetic acid solution flows through six-way injection valve (2), to be written into high performance liquid chromatography separating column (3) by the testing sample that syringe (6) are injected in the six-way injection valve (2), enter online reduction column (4) after each seed amino acid in the sample is separated, amino acid disulfide in the solution is reduced into corresponding mercaptoamino acid by the TCEP complex compound, again successively with triangle gold nano solution, luminol solution, superoxol on-line mixing, enter at last chemiluminescence detector (5) and carry out on-line determination;
Described testing sample is human plasma or urine, wherein contained amino acid is the potpourri of mercaptoamino acid and amino acid disulfide, and wherein mercaptoamino acid is the potpourri of halfcystine, cysteinyl glycine, homocysteine, glutathione and glutamyl cysteine; Amino acid disulfide is the potpourri of cystine and homocystine;
Described triangle gold nano solution concentration is 0.2-1.06nmol/L;
The trifluoroacetic acid percent by volume is that 0.05-0.25%, luminol solution volumetric molar concentration are that 0.002-0.02mmol/L, superoxol volumetric molar concentration are 0.2-10mmol/L in the described trifluoroacetic acid solution;
Described trifluoroacetic acid solution, triangle gold nano solution, luminol solution, the flow velocity of superoxol in system are 0.2-2mL/min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105866300A (en) * | 2016-06-15 | 2016-08-17 | 博莱克科技（武汉）有限公司 | Determination method of amino metabolites |
| CN106443028A (en) * | 2016-08-02 | 2017-02-22 | 上海海洋大学 | Method for simultaneously measuring glutathione and free amino acid in shellfish |
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| CN101158691A (en) * | 2007-11-16 | 2008-04-09 | 东南大学 | Electrochemistry detecting method and testing apparatus of saccharification hemoglobin content |
| CN102590187A (en) * | 2011-01-12 | 2012-07-18 | 北京化工大学 | Analysis method for using magnesium-aluminium carbonate hydrotalcite to catalyze luminol-hydrogen peroxide chemiluminescence |
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Patent Citations (2)
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| CN101158691A (en) * | 2007-11-16 | 2008-04-09 | 东南大学 | Electrochemistry detecting method and testing apparatus of saccharification hemoglobin content |
| CN102590187A (en) * | 2011-01-12 | 2012-07-18 | 北京化工大学 | Analysis method for using magnesium-aluminium carbonate hydrotalcite to catalyze luminol-hydrogen peroxide chemiluminescence |
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|---|
| GEOFFREY P. MCDERMOTT 等: "Direct Detection of Biologically Significant Thiols and Disulfides with Manganese(IV) Chemiluminescence", 《ANALYTICAL CHEMISTRY》, 6 July 2011 (2011-07-06) * |
| QINGSHUO CHEN 等: "The new approach for captopril detection employing triangular gold nanoparticles-catalyzed luminol chemiluminescence", 《TALANTA》, 8 December 2011 (2011-12-08) * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105866300A (en) * | 2016-06-15 | 2016-08-17 | 博莱克科技（武汉）有限公司 | Determination method of amino metabolites |
| CN106443028A (en) * | 2016-08-02 | 2017-02-22 | 上海海洋大学 | Method for simultaneously measuring glutathione and free amino acid in shellfish |
| CN106443028B (en) * | 2016-08-02 | 2018-07-27 | 上海海洋大学 | Method that is a kind of while measuring shellfish GSH-PX activity and free amino acid |
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